Respiratory and Sleep Medicine, Women's and Children's Hospital, North Adelaide, Australia.
Adelaide Medical School, University of Adelaide, Adelaide, Australia.
Hum Gene Ther. 2021 Aug;32(15-16):817-827. doi: 10.1089/hum.2021.031. Epub 2021 Jun 24.
A gene addition therapy into the conducting airway epithelium is a potential cure for cystic fibrosis lung disease. Achieving sustained lung gene expression has proven difficult due to the natural barriers of the lung. The development of lentiviral (LV) vectors pseudotyped with viral envelopes that have a natural tropism to the airway has enabled persistent gene expression to be achieved . The aims of this study were to compare the yields of hemagglutinin (HA) and vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped HIV-1 vectors produced under the same conditions by our standard LV vector production method. We then sought to measure gene expression in mouse airways and to determine whether lysophosphatidylcholine (LPC) conditioning enhances short- and long-term gene expression. C57Bl/6 mouse airways were conditioned with 10 μL of 0.1% LPC or saline control, followed 1 h later by a 30 μL dose of an HA or VSV-G pseudotyped vector carrying either the or reporter genes. LacZ expression was assessed by X-gal staining after 7 days, while lung luminescence was quantified regularly for up to 18 months by bioluminescent imaging. The HA pseudotyped vectors had functional titers 25 to 60 times lower than the VSV-G pseudotyped vectors. Conditioning the lung with LPC significantly increased the total number of LacZ-transduced cells for both pseudotypes compared to saline control. Regardless of LPC conditioning, the VSV-G pseudotype produced higher initial levels of gene expression compared to HA. LPC conditioning did not increase the number of transduced basal cells for either pseudotype compared to saline, and was not required for long-term gene expression. Both pseudotyped vectors effectively transduced the upper conducting airways of wild-type mice. The use of LPC conditioning before vector delivery was not required in mouse lungs to produce long-term gene expression, but did improve short-term gene expression.
气道上皮的基因添加疗法是囊性纤维化肺病的潜在治疗方法。由于肺部的天然屏障，实现持续的肺部基因表达一直很困难。开发具有气道天然趋向性的包膜假型慢病毒 (LV) 载体，使持续的基因表达得以实现。本研究的目的是比较在相同条件下，使用我们的标准 LV 载体生产方法生产的血凝素 (HA) 和水疱性口炎病毒糖蛋白 (VSV-G) 假型 HIV-1 载体的产量。然后，我们试图测量小鼠气道中的基因表达，并确定溶血磷脂酰胆碱 (LPC) 调理是否增强短期和长期基因表达。用 10μL0.1% LPC 或盐水对照调理 C57Bl/6 小鼠气道，1 小时后用携带或报告基因的 HA 或 VSV-G 假型载体给予 30μL 剂量。7 天后通过 X-gal 染色评估 LacZ 表达，而通过生物发光成像定期定量检测长达 18 个月的肺发光。HA 假型载体的功能滴度比 VSV-G 假型载体低 25 到 60 倍。与盐水对照相比，用 LPC 调理肺部可显著增加两种假型的 LacZ 转导细胞总数。无论 LPC 调理与否，与 HA 相比，VSV-G 假型都产生更高的初始基因表达水平。与盐水相比，LPC 调理不会增加两种假型的转导基底细胞数量，也不需要长期基因表达。两种假型载体都有效地转导了野生型小鼠的上呼吸道。在小鼠肺部中，在载体递送前使用 LPC 调理不是产生长期基因表达所必需的，但确实可以改善短期基因表达。
